16S rRNA sequencing raw data from a thermophilic trickle bed reactor for biogas upgrading

The experiment was conducted in the biogas laboratory at the Norwegian Institute of Bioeconomy Research (NIBIO) (1430 Ås, Norway). The inoculum used was collected from manure-based lab-scale continuous stirring tank reactors (CSTRs) with a working volume of 15 L, which were operated at a temperature of 51 ºC. After collection, the inoculum was filtered using a sieve with diameter of 0.212 mm to remove solid particles. Following filtration, the total solids (TS%) and volatile solids (VS%) in the filtered inoculum were measured at 3.00 ± 0.01 and 1.82 ± 0.01%, respectively, based on the method described by APHA (2017). The pH of the filtered inoculum was 7.89. The filtered inoculum then underwent two-week of degassing procedure in a shaking incubator at 90 rpm to remove residual organic matter at both mesophilic (37 ºC) and thermophilic (51 °C) temperatures. The degassed inoculum was then mixed with 10% (v/v) nutrient solution. This solution contained 408×10−3g/L KH2PO4, 426×10−3g/L Na2HPO4, 110×10−3g/L CaCl2×2H2O, 100×10−3g/L MgCl2×6H2O, 300×10−3 g/L NH4Cl, 300×10−3g/L NaCl and 1 mL/L of trace elements. The trace elements were prepared by mixing 1500 mg/L FeCl2. 4H2O, 70 mg/L ZnCl2, 100 mg/L MnCl2 × 4H2O, 6 mg/L H3BO3, 190 mg/L CoCl2 × 6H2O, 2 mg/L CuCl2 × 2H2O, 24 mg/L NiCl2×6H2O and 36 mg/L Na2MoO4 × 2H2O.
Two laboratory-scale TBRs with a working volume of 815 mL were utilized to further investigate the impact of H2S on biomethanation. One of the TBR is assigned as control (TBR-Control) and the other as test (TBR-H2S). The TBRs were filled with 145g of polyethylene biocarriers CFAS® (surface area=650 m2/m3) (Biowater Technology AS, Norway). The TBRs were inoculated with 150 mL of degassed inoculum (containing 10% v/v nutrients solution) and supplied with biogas with a mixture of CH4, CO2, and H2. The gas injection was done at the bottom of the reactor in a counter-current direction through a fish stone diffuser with a gas loading of 3.68 L/Lreactor. d in 4:1 H2 to CO2 ratio, respectively. The substrate gas flow was controlled by a mass flow controller (MC 50, Alicat Scientific, Tucson, AZ 85743, U.S.A.). The recirculation of inoculum was carried out continuously at a rate of 60 mL/min using a peristaltic pump (530 DU, Watson-Marlow Limited, UK). The reactors were maintained at a thermophilic temperature of 51 ºC by recirculating water through the reactor water jacket from a water bath (CORIO CD-BC4 heating circulator, JULABO GmbH, Germany). The temperature of the room was continuously monitored using a digital thermometer attached to near the TBRs ((EL-WiFi-TP Wireless Temperature Data Loggers, Ohio, U.SA.). The experiment lasted 220 days and every week 30 mL of liquid was removed and replaced with the same volume of new degassed inoculum to provide a continuous source of nutrients for the microbes and prevent buildup of inhibitory compounds. Excess water generated during the process was also removed to maintain the same level of liquids throughout the experiment.
The experiment was conducted in two consecutive periods (Period-I and II). The purpose of Period-I was to acclimatize the methanogens inside the inoculum with a mixture of the substrate gases (containing H2, CH4 and CO2) and promote them to produce stable CH4 content in both reactors before the commencement of H2S experiment. The Period-II was specifically designed to assess the effect of H2S on biomethanation and microbial community structure. The TBR experiment was conducted in two periods as described below:
Period I (adaptation period): During this period, both TBRs were supplied and acclimatized with H2 and biogas containing 60% CH4 and 40% CO2 (H2: CO2 ratio = 4:1). The gas loading was 3.68 L/ Lreactor.d and the gas retention time (GRT) was 6.52 hrs. The substrate gas composition injected after mixing during this period was 62% H2, 15% CO2 and 23% CH4. This period aimed to facilitate the formation of biofilm on the packing material and to acclimate to the input gas. The adaptation period (Period I) lasted 120 days. The average normalized CH4 content (%) was considered stabilized when it remained constant for 8-10 consecutive days, signalling the beginning of the test period (Period II). The average CH4 content and yield data for this period are included in this study.
Period II (experimental period): During this period, TBR-Control continued with the same gas composition and loading as in Period I. TBR-H2S was injected with H2 and H2S-rich biogas containing 57% CH4, 40% CO2, and 3% H2S (H2: CO2 ratio = 4:1), with a gas loading of 3.68 L/Lreactor.d and GRT of 6.52 hrs. The gas composition injected into TBR-H2S in this period was 61.54% H2, 15.38% CO2, 21.92% CH4, and 1.15% H2S. The experimental period (Period II) lasted 100 days. During Period II, the gas volume from TBR-H2S could not be accurately measured due to safety concerns (high H2S content from the effluent gas, over 13,000ppm). Consequently, the performance of TBR-H2S was assessed based on the composition of the effluent gas. The effluent gas volume was assumed to be like that of TBR-Control, given that the input gas loading and composition were comparable.
Samples for microbial analysis were stored in 2 mL Eppendorf tubes at -18ºC to preserve microbial integrity (Poulsen et al., 2021). One sample was taken from the adaptation period, and four samples (Day 4, 32, 46, 60) were taken during the experimental period (Day 4, 32, 46, 60). Microbial community analysis was conducted by DNASense (DNASense, Niels Jernes Vej 10, 9220 Aalborg, Denmark). The DNA extraction followed by method adapted from Jensen et al. (2023), utilizing the DNeasy 96 PowerSoil Pro QIAcube HT Kit. Each sample was mixed with 500 μL of CD1 solution and subjected to bead beating at 1600 rpm for two minutes, repeated three times with breaks of two minutes on ice in between. Following lysis, samples were centrifuged at 3500 x g for ten minutes. The supernatant of each sample was transferred to a new S-block well and mixed with CD2 and nuclease-free water in a 3:3:1 (v/v) ratio. After mixing, the samples were centrifuged again at 3500 x g for 10 minutes, and the process continued accordingly. Analysis and reporting were performed using DNASense’s custom bioinformatic workflow (version MCA_ONT_DS230723). Amplicon libraries for the 16S/18S rRNA gene variable regions 48 (abeV48A) were prepared using a custom protocol. Up to 25 ng of extracted DNA was used as a template for PCR amplification. Each PCR reaction (50 μL) contained 0.2 mM dNTP mix, 0.01 units of Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific, U.S.A.), and 500 nM of each forward and reverse primer in the supplied SuperFI Buffer. PCR was done with the following program: Initial denaturation at 98 °C for 3 min, 25 cycles of amplification (98 ºC for 30 s, 62 ºC for 20 s, 72 ºC for 2 min), and a final elongation at 72 ºC for 5 min. The forward and reverse primers used include custom 24 nt barcode sequences followed by the sequences targeting abeV48A: [515FB] GTGYCAGCMGCCGCGGTAA and [1391R] GACGGGCGGTGWGTRCA (Apprill et al., 2015; Parada et al., 2016). The resulting amplicon libraries were purified using the standard protocol for CleanNGS SPRI beads (CleanNA, NL) with a bead-to-sample ratio of 3:5. Subsequently, DNA was eluted in 25 μL of nuclease free water (Qiagen, Germany). Sequencing libraries were then prepared from the purified amplicon libraries using the SQKLSK114 kit (Oxford Nanopore Technologies, UK) following the manufacturer’s protocol with the following modifications: 500 ng total DNA was used as input, and CleanNGS SPRI beads were employed for library cleanup steps. DNA concentration was measured using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, U.S.A.). A subset of amplicon libraries underwent validation for product size and purity through Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes (Agilent, U.S.A.). The resulting sequencing library was loaded onto a MinION R10.4.1 flowcell and sequenced using the MinKNOW 23.04.6 software (Oxford Nanopore Technologies, UK). Reads were basecalled and demultiplexed with MinKNOW guppy g6.5.7 using the super accurate basecalling algorithm (config r10.4.1_400bps_sup.cfg) and custom barcodes.The sequencing reads in the demultiplexed and basecalled fastq files were filtered for length (320-2000 bp) and quality (phred score > 15) using a local implementation of filtlong v0.2.1 with the settings –min_length 320 –max_length 2000 –min_mean_q 97. The filtered reads were mapped to the QIIME formatted MiDAS database, release 4.8.1 with minimap2 v2.24-r1122 using the -ax -mapont command and downstream processing using samtools v1.14. Potential generic place holders and deadend taxonomic entries were cleared from the taxonomy flat file, i.e. entries containing uncultured, metagenome or unassigned, were replaced with a blank entry. Mapping results were filtered such that query sequence length relative to alignment length deviated < 5 % and alignment mapping quality > 0.79. Noteworthy, for both data sets lowabundant OTUs making up < 0.01 % of the total mapped reads within each sample were disregarded as a data denoisingstep. Further bioinformatic processing was done via RStudio IDE (2023.3.0.386) running R version 4.3.2 (20231031) and using the R packages: ampvis2 (2.8) [10], tidyverse (2.0.0), seqinr (4.2.30), ShortRead (1.58.0) andiNEXT (3.0.0).